EP2591776B1 - Use of kukoamine a and kukoamine b - Google Patents

Use of kukoamine a and kukoamine b Download PDF

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EP2591776B1
EP2591776B1 EP11774262.7A EP11774262A EP2591776B1 EP 2591776 B1 EP2591776 B1 EP 2591776B1 EP 11774262 A EP11774262 A EP 11774262A EP 2591776 B1 EP2591776 B1 EP 2591776B1
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lps
cpg dna
cells
shows
tnf
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French (fr)
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EP2591776A4 (en
EP2591776A1 (en
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Jiang Zheng
Xin Liu
Xinchuan Zheng
Hong Zhou
Hongwei Cao
Ning Wang
Yongling Lu
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First Affiliated Hospital of TMMU
Tianjin Chase Sun Pharmaceutical Co Ltd
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First Affiliated Hospital of TMMU
Tianjin Chase Sun Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A61K36/815—Lycium (desert-thorn)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to use of Kukoamine A and Kukoamine B in the preparation of drugs for the prevention and treatment of sepsis and autoimmune disease caused by bacteria.
  • Sepsis and autoimmune disease are caused by body's excessive immune response, to which there were no reliable and effective drug strategies yet.
  • Sepsis is an acute systemic inflammatory response syndrome, the mortality rate of which could be as high as 30%-70%, and it has been a serious threat to critically ill patients.
  • Autoimmune disease is a chronic inflammation disease.
  • the incidence rate of autoimmune disease is about 3.2%-5.3% in China, and it has been one of the leading causes of death of women under 65. Therefore, appropriate prevention and treatment measures for sepsis and autoimmune disease have been a hot spot for clinical research.
  • LPS bacterial endotoxin/ lipopolysaccharide
  • CpG DNA unmethylated DNA
  • LPS and CpG DNA In the course of acute infection, rapid invasion of pathogens may generate large amount of LPS and CpG DNA, which induce large number of expression and release of various inflammatory mediators, such as TNF- ⁇ , IL-1 ⁇ , IL-6 etc., within a short priod of time, leading to early organ dysfunction and late immune paralysis, and resulting in death of sepsis patients.
  • various inflammatory mediators such as TNF- ⁇ , IL-1 ⁇ , IL-6 etc.
  • TNF- ⁇ , IL-1 ⁇ , IL-6 etc. the persistence of LPS and CpG DNA may lead to persistance and chronic evolution of inflammatory reaction, and induce mass production of inflammatory mediators, immunoglobulins and rheumatoid factor etc., which form immune complex depositing on synovium, activate complement, product anaphylatoxin, resulting in inflammatory pathologic damage, and eventually leading to organ injury.
  • LPS and CpG DNA could prevent and cure sepsis and autoimmune disease from the source.
  • Related drug researches have confirmed that the blockade of irritant reaction of LPS and CpG DNA to immunocyte and the inhibition of release of inflammatory mediators, like TNF- ⁇ , have obvious therapeutic effects on sepsis and autoimmune disease. Therefore, antagonistic activity on LPS and CpG DNA could reflect preventive and therapeutic effects of certain drugs to sepsis and autoimmune disease.
  • Thunb., Forsythia suspensa (Thunb.) Vahl, Scutellaria baicalensis Georgi, Artemisia annua L. and other more than 20 kinds of Chinese herbal medicines have good antagonistic activity on pathogen-associated molecular patterns, e.
  • decoction of Atractylodis macrocephalae rhizome can significantly reduce the elevated levels of LPS and TNF- ⁇ in serum of patients with rheumatoid arthritis, suppress the levels of IgG, IgA and IgM in serum, reduce RF (rheumatoid factor) positive rate, and thus achieve treatment of rheumatoid arthritis; Langchuang formula, which consists of 7 kinds of Chinese herbal medicines for heat-clearing and detoxicating, such as Oldetilandia diffusa (Willd.) Roxb., Scutellaria barbata D.
  • Lycii cortex is the dried root bark of Lycium chinense Mill. or Lycium barbarum L of the Solanaceae family. In traditional Chinese medicine theory, it has the effects of clearing away the heat-evil and expelling superficial evils. Modern pharmacological studies have found that Lycii vortex contains alkaloids, organic acids, anthraquinones and peptides, and it plays a pharmacological role of anti-hypertension, anti-hyperglycemia, relieving fever and analgesia. However, researches and applications on Lycii cortex antagonizing LPS and CpG DNA and treating sepsis and autoimmune disease have not been reported in domestic and foreign literatures and domestic invention patents up to now.
  • Gajanan Sherbet British Journal of Medical Practitioners, 2009:2(1), p. 6-13 reviews bacterial infections as cause for autoimmune diseases via upregulation of toll-like receptors (TLR) and nitric oxide synthase (NOS).
  • TLR toll-like receptors
  • NOS nitric oxide synthase
  • Lee et al. Journal of Ethnopharmacology 124(3): 530-538 (2009 ) discloses a Chinese medicine comprising an extract of 4 raw herbs including cortex lyci, which extract was shown to inhibit inflammation in lung tissue in response to LPS.
  • the purpose of present invention is to overcome the major defect of uncertainty of material basis and mechanisms of constituents and extractives in traditional Chinese medicine, develop a safe, effective, quality controllable drug for prevention and treatment of sepsis and autoimmune disease, and solve the lack of effective drugs in clinical treatment at present.
  • the technical solution of present invention is:
  • Said Kukoamine A and Kukoamine B are extracted from Lycii cortex in traditional Chinese medicine.
  • Said drug is used for antagonizing the key factors, i.e. bacterial endotoxin/ lipopolysaccharide (LPS) and unmethylated DNA of bacteria, which induce the development of sepsis and autoimmune disease caused by bacteria.
  • LPS bacterial endotoxin/ lipopolysaccharide
  • Said Lycii cortex is the dried root bark of Lycium chinense Mill. or Lycium barbarum L. of the Solanaceae family.
  • the applicant persists for a long time in drug research of antagonizing pathogen-associated molecular patterns targeting at LPS and CpG DNA.
  • biosensor screening and tracking platform established, the applicant screens and directionally isolates the activated monomer, which has effects of binding and antagonizing LPS and CpG DNA, from traditional Chinese medicine for clearing away the heat-evil and expelling superficial evils.
  • the present invention uses Lycii vortex as raw material, establishes drug screening and directional separating platform targeting at lipid A and CpG DNA, and uses binding activity of Chinese herb extracts with lipid A and CpG DNA as screening indicators.
  • Raw material was boiled with water; then, it was extracted, isolated and purified respectively with methods of macroporous adsorptive resins, cation exchange and reversed-phase high-performance liquid chromatography; through pharmacological evaluation in vivo and in vitro, such as experiments of neutralization of LPS in vitro, experiments of inhibition on bindings of LPS and CpG DNA with cells, experiments of inhibition on inflammatory reaction caused by stimulations of LPS and CpG DNA, and experiments of protection of model animal with sepsis, eventually, two active constituents, Kukoamine A (KA) and Kukoamine B (KB), which have good antagonistic effect on LPS and CpG DNA, were screened out.
  • KA Kukoamine A
  • KB Kukoamine B
  • the present invention separates active constituents antagonizing LPS and CpG DNA from traditional Chinese herbs, and provides safe, reliable and effective drugs for prevention and treatment of sepsis and autoimmune disease.
  • Said Kukoamine A and Kukoamine B are spermine-like alkaloids extracted from Lycii cortex. They are a pair of isomeride, with the same molecular formula of C 28 H 42 N 4 O 6 and molecular weight of 530.66. Their chemical structures are respectively as follows:
  • kukoamine A and Kukoamine B for use of the present invention have a brilliant prospect of being effective drugs in treatment of sepsis and autoimmune disease.
  • Kukoamine A and Kukoamine B have high affinity with LPS and CpG DNA, can significantly neutralize LPS and CpG DNA, blockade their bindings with RAW264.7 cells (murine macrophages), inhibit the expression and release of inflammatory mediators (TNF- ⁇ , IL-6, etc.) in RAW264.7 cells induced by LPS and CpG DNA, and finally blockade the inflammatory activation of cells and prevent disorders of the immune response.
  • Kukoamine A and Kukoamine B can lower the levels of LPS and TNF- ⁇ in mice injected with heat-killed Escherichia coli (mimic vivo injection of LPS and CpG DNA), play a role in antagonism on LPS and CpG DNA, and improve survival rates of the mice.
  • LPS and CpG DNA are key factors inducing sepsis and autoimmune disease
  • antagonistic activity on LPS and CpG DNA can reflect preventive and therapeutic effects of certain drugs to sepsis and autoimmune disease.
  • the research models selected in the detailed description are all used to evaluate the binding and antagonistic activity of KA and KB on LPS and CpG DNA, and to reflect treatment effect of KA and KB to sepsis and autoimmune disease.
  • the present invention will be further descripted through following embodiments. It should be pointed out that the following embodiments are intended to illustrate rather than limit the disclosure.
  • Table 1 Name and origin of 114 kinds of traditional Chinese herbs Latin name Origin Latin name Origin Latin name Origin Holboellia latifolia Wall. Sichuan province Cinnamomum cassia Presl Guangxi City Fraxinus rhynchophylla Hance Sichuan City Patrinia scabiosaefolia Fisch. Sichuan province Piper kadsura (Choisy) Ohwi Zhejiang province Gentiana macrophylla Pall. Gansu province Lobelia chinensis Lour. Sichuan province Nelumbo nucifera Gaertn. Sichuan province Artemisia annua L. Hubei province Scutellaria barbata D.
  • Absolute ethyl alcohol (EtOH) and disodium hydrogen phosphate (Na 2 HPO 4 ) were purchased from Chongqing Chuandong (Group) Chemical Factory Co. Ltd.; AB-8 Macroporous adsorption resin and D001 cation exchange gel column were purchased from Chemical Plant of NanKai University in Tianjin; Trifluoroacetic acid (TFA) was purchased from Tianjin Guangfu Fine Chemical Research Institute; methanol (MeOH) was purchased from Hyclone company (USA); GIBCO®DMEM culture medium was purchased from Invitrogen company (USA); fetal calf serum (NCS) was purchased from Hyclone company (USA); PBS (20 mM, pH 7.2) was purchased from WuHan Boster Biological Technology., LTD; hydrochloric acid (HCl) was purchased from Chongqing Chuandong chemical plant (Group) Co., Ltd.; RAW264.7 cells and reference strain Escherichia coli.
  • Immobilization of lipid A and CpG DNA Using Optically-Based Affinity Biosensors technology, Lipid A, the biologic active centres of LPS, and CpG DNA were respectively, immobilized on the reacting surfaces of cuvettes in an IAsys plus affinity biosensor according to the manufacturer's instructions of IAsys Affinity Sensor.
  • the end of hydrophobic side chain of lipid A was immobilized on cuvette with hydrophobic surface, and the phosphate group (active group of lipid A) at the other end was floating and exposing to the outside, which act as target spot of binding with active constituents for disease treatment in traditional Chinese medicine.
  • the active constituents in solution were binded to lipid A through electrostatic interaction.
  • Biotinylated CpG DNA was immobilized on the surface of a biotin cuvette by linking to avidin, which had been coated on the surface of a biotin cuvette, and then the unlabeled group of CpG DNA was floating and exposing to the outside, which act as target spot of binding with active constituents for disease treatment in traditional Chinese medicine.
  • the active constituents in solution were binded to CpG DNA through the electrostatic interaction and embedment.
  • Detection of the aqueous herbal extract The crude drugs of 114 traditional Chinese herbs were pulverized. 1 g of each powder was added with 10 ml distilled water, boiled at 100 °C for 1.5 h, centrifuged at 4000 rpm for 20 min, filtrated and then collected the supernatant. 5 ⁇ l of aqueous herbal extracts of each herb were used to detect their binding with lipid A or CpG DNA.
  • 1Binding reaction 45 ⁇ l PBS was added in cuvette; then 5 ⁇ l samples was added in cuvette; liquid were draw-out after binding response reach a plateau; 2Dissociation: Cuvette was washed thrice in 50 ⁇ l PBS; liquid were draw-out after dissociation reach a plateau; 3 Regeneration: Cuvette was washed thrice in 50 ⁇ l 0.1 N HCl; liquid were draw-out after regeneration reach a plateau; 4 Cuvette was washed thrice in 50 ⁇ l PBS; next cycle was started to detect new sample after curves back to baseline levels and smoothed. Data analysis was performed using the FASTplot software after detection finished.
  • Lycii cortex has high affinity with both lipid A and CpG DNA, among which, Lycii cortex has the highest affinity.
  • the results suggest that Lycii cortex has greater potential of containing active constituents antagonizing LPS and CpG DNA than other traditional Chinese herbs, so it was selected as study object of extraction and separation. The results were shown in Figure 1 .
  • Figure 1 a is a response curve of immobilization of lipid A
  • Figure 1b is a response curve of immobilization of CpG DNA
  • Figure 1c shows binding reaction of lipid A with Lycii cortex and other 6 kinds of traditional Chinese herbs
  • Figure 1d shows binding reaction of CpG DNA with Lycii cortex and other 6 kinds of traditional Chinese herbs.
  • CL-4 lyophilization powder was diluted into ultrapure water to make 100 mg/ml solution, filtered by a 0.45 ⁇ m filter membrane, loaded onto D001 cationic exchange gel column, and eluted with distilled water, 0.3 M Na2HPO4 and 0.5 M Na2HPO4 successively. The eluted fractions were separately collected, and lyophilized after concentrated under reduced pressure. Three constituents were obtained, and named CL-4a, -4b and -4c according to their elution order. CL-4 constituents were separately dissolved in PBS to make 1.0 mg/ml solution, 5 ⁇ l of which were loaded. Their binding activity with lipid A and CpG DNA were detected according to methods of embodiment 1.
  • CL-4b was dissolved into LPS-free water to make 8 ⁇ g/ml solution, and incubated with equal volume of LPS (1 ng/mL) at 37°C for 30 min. Subsequently, 100 ⁇ L mixed solution of CL-4b and LPS was added with equal volume of the quantitative TAL reagents dissolved in LPS-free water, gently shaked to mix the contents, and reacted at 37°C for 60 min in kinetic tube reader. The agglutination of TAL reagent induced by the existence of non-neutralized LPS was measured. The mixture of LPS and equal volume of LPS-free water act as positive control. Each group contained three repeated tubes. The result was expressed in EU/ml, the endotoxin unit of LPS. Operation was performed according to the manufacturer's instructions of EDS-99 Bacterial Endotoxin Detecting system.
  • Cl-4b can not lead to TAL agglutination by itself, but it can significantly reduce the agglutination induced by LPS after incubated with LPS for 30 min. The result suggests that CL-4b has neutralizing activity on LPS. The results were shown in Figure 4 .
  • RAW 264.7 cells were adjusted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% NCS (v/v), transferred into 96-well plate (200 ⁇ l per well), cultured at 37 °C in a 5% CO2 humidified incubator for 4 h, and loaded after cells attachment; for the purpose of the experiment three groups were established: medium group, stimulation group and drug treatment group, and each group contained three repeated wells; medium group was added with no reagent; stimulation group was added with LPS (final concentration of 100 ng/ml) or CpG DNA (final concentration of 10 ⁇ g/ml); drug treatment group was added with CL-4b (final concentration of 200 ⁇ g/ml), as well as LPS (final concentration of 100 ng/ml) or CpG DNA (final concentration of 10 ⁇ g/ml); cells were cultured at 37 °C in a 5% CO2 humidified incubator for 24 h, and the supernatant was collected for further detection. Detections of TNF- ⁇ and IL-6 were performed
  • CL-4b does not induce the release of TNF- ⁇ and IL-6 in RAW264.7 cell by itself, but it can significantly reduce the release of of TNF- ⁇ and IL-6 in RAW264.7 cell induced by LPS and CpG DNA.
  • Figure 5 shows the inhibition of CL-4b on the release of TNF- ⁇ and IL-6 in RAW264.7 cells induced by LPS
  • Figure 5b shows the inhibition of CL-4b on the release of TNF- ⁇ and IL-6 in RAW264.7 cells induced by CpG DNA.
  • E. coli heat-killed Escherichia coli ATCC 35218
  • Bacteria culture was performed according to Clinical Laboratory Procedures. Single bacterial colony of E. coli from LB agar plates were picked and transferred into 10 mL sterile liquid of LB broth using a sterile inoculating loop, and cultivated at 37°C in a shaker (250 rpm). After medium become turbid, these culture medium were then transferred to 2000 mL of fresh LB medium and cultivated at 37°C in a shaker (250 rpm) for 12 h. The suspension was collected, transferred into 1000 ml centrifuge tube, and centrifuged at 5000 rpm for 15 min. The supernatant was discarded.
  • the bacteria were collected, washed and resuspended into sterile saline, and then centrifuged again under the same condition. Above-mentioned process was repeated thrice.
  • the bacteria pellet was resuspended with a pipet in 50 ml sterile saline, transferred into 100 ml saline bottle, and boiled in electric furnace for 30 min. Then the suspension of heat-killed Escherichia coli was obtained.
  • the suspension of E. coli was diluted by 100 fold, and measured OD value at 600 nm on nucleic acid protein analyzer. Conversion was made according to the regression equation of OD value and concentration, and the suspension was diluted according to the conversion result. Then the operating fluids for mice injection were obtained.
  • CL-4b control group was injected with CL-4b (60 mg/kg) and sterile saline; heat-killed E. coli group was injected with heat-killed Escherichia coli (1.0 ⁇ 10 10 CFU/ml) and sterile saline; CL-4b treatment group was injected with CL-4 (60 mg/ml) at 10 min after heat-killed E.
  • mice were coli injection.
  • the injection volum of each solution was 200 ⁇ l per 20 g body weight via tail vein.
  • Injection volume of each mouse was 200 ⁇ l per 20 g body weight.
  • the general status and mortality rate of mice were observed for 7 days, and survival differences between CL-4b control group and CL-4b treatment group were compared.
  • CL-4b 1 and CL-4b 2 were analyzed by UV spectrum, IR spectrum, NMR spectrum and mass spectrum.
  • CL-4b 1 and CL-4b 2 are both pale yellow crystals.
  • UV spectrum detection shows that the absorption peaks ( ⁇ max ) of the two components are both at point 281 nm (methanol); and results of ESI-MS of the two components are both [M+H] + m/z 531, which suggests that they are a pair of isomeride.
  • CL-4b 1 was identified as Kukoamine A (KA)
  • CL-4b 2 was identified as Kukoamine B (KB).
  • KA and KB were separately dissolved to make 100 ⁇ M operating solution; 5 ⁇ l of the solution was loaded; their binding activity with lipid A and CpG DNA were detected according to methods of embodiment 1.
  • KA and KB both have high affinity with lipid A and CpG DNA.
  • the results were shown in Figure 8 .
  • Figure 8a shows the affinity detection of KA and KB with lipid A
  • Figure 8b shows the affinity detection of KA and KB with CpG DNA.
  • KA and KB(1, 2 and 4 ⁇ g/ml) were separately mixed with equal volume of LPS(2.0 ng/ml) and pre-incubated at 37°C for 30 min; LPS control group was added with equal volume of nonpyrogenic water; LPS value was detected by kinetic turbidimetric limulus test after incubation; detection of each concentration was repeated three times; LPS content was expressed by means of mean ⁇ standard deviation; operation was performed according to the manufacturer's instructions of EDS-99 Bacterial Endotoxin Detecting system.
  • RAW264.7 cells were diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10%(v/v) NCS, added into 24-well cell culture plates, cultured at 37 °C in a 5% CO2 humidified incubator for 4 h, added with KA and KB(0, 200 ⁇ g/ml) after cells attachment, and then added with FITC-labeled LPS(final concentration of 400 ng/ml) and 5-FAM CpG DNA(final concentration of 10 ⁇ g /ml); at the same time, medium group without any reagent was established; subsequently, RAW264.7 cells were incubated for 30 min, washed thrice in PBS, resuspended with a pipet and transferred into EP tube, and immobilized with 4% paraform for 10 min; then cells were washed thrice in PBS, made into cell suspension, and detected by flow cytometry; the detections of each group were repeated thrice. Mean fluorescence intensity was expressed by means of Mean ⁇ standard
  • KA and KB can significantly decrease the fluorescence intensity of LPS and CpG DNA (p ⁇ 0.01) in RAW264.7 cells, which suggested that they can influence the binding of LPS and CpG DNA with RAW264.7 cells, effectively inhibite the excessive immune response induced by LPS and CpG DNA, prevent injury to the body, and thus play a role in the prevention and treatment of sepsis and autoimmune disease.
  • the results were shown in Figure 10 .
  • Figure 10a shows the influence of KA and KB on the binding of fluorescently-labeled LPS with RAW264.7 cells
  • Figure 10b shows the influence of KA and KB on the binding of fluorescently-labeled CpG DNA with RAW264.7 cells.
  • RAW264.7 cells were diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS, added into 96-well plates (200 ⁇ l per well), and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h until cells adhere to the wall; culture medium were replaced with fresh culture medium, added with KA and KB (final concentrations of 0, 50 and 100 ng/ml), and added with LPS (final concentration of 100 ng/ml) and CpG DNA (final concentration of 10 ⁇ g/ml); at the same time, medium group without any reagent was established; subsequently, RAW264.7 cells were incubated for 4 h; and the supernatant was collected for further detection; dectection of the TNF- ⁇ concentration was performed according to the manufacturer's instructions of ELISA kit. Result was expressed by means of mean ⁇ standard deviation.
  • KA and KB can inhibit the release of TNF- ⁇ induced by LPS and CpG DNA in a dose-dependent manner, which has significant difference compared with control group (p ⁇ 0.01). Extended release of a large amount of TNF- ⁇ play an important role in the pathological damage in sepsis and autoimmune disease, therefore inhibition on TNF- ⁇ release can effectively prevent and cure sepsis and autoimmune disease.
  • Figure 11 a shows the influence of KA and KB on the release of TNF- ⁇ in RAW264.7 cells induced by LPS
  • Figure 11b shows the influence of KA and KB on the release of TNF- ⁇ in RAW264.7 cells induced by CpG DNA.
  • MTT assay were adoped for cell vitality detection; RAW264.7 cells were diluted to 1 ⁇ 10 6 /ml in DMEM medium, added into 96-well plates(200 ⁇ l per well), and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h; treatment group was successively added with KA and KB (final concentration of 200 ⁇ g/ml); no reagent was added in medium group; each group has 6 parallel wells; subsequently, RAW264.7 cells were cultured for 24 h; the supernatant was discarded; each well was added with 180 ⁇ l culture medium and 20 ⁇ l MTT solution(5mg/ml), and cultured for 4 h; the supernatant was removed; 150 ⁇ l of dimethyl sulphoxide was added into each well; the 96-well plates were shook for 10 min for the dissolution of the crystals; RAW264.7 cells vitality was expressed as absorbance values at 550 nm (OD550) of each well; the
  • Supernatant of heat-killed E. coli was prepared according to 2.3.3 items of embodiment 2; absorbance values of the supernatant were assayed at 600 nm (OD 600 value 1.0 ⁇ 1.0 ⁇ 10 10 CFU/ml); a total of 84 Kunming mice, half male and half female, were divided into three groups randomly: heat-killed E. coli control group, KA (40 mg/kg) plus heat-killed E. coli group and KB (40 mg/kg) plus heat-killed E. coli group; each group has 28 mice; after animals were weighed, heat-killed E. coli control group was injected with heat-killed E.
  • E. coli(1.1 ⁇ 10 10 CFU/ Kg) the other two groups were respectively injected with KA or KB (40 mg/kg) at 10 min after injection of heat-killed E. coli(1.1 ⁇ 10 10 CFU/ Kg); the total injection of each animal was 200 ⁇ l per 20 g body weight; orbital venous blood of mice was collected at time point of 0, 2, 4, 8, 12, 24, 48 and 72h after injection; LPS level was assayed by kinetic turbidimetric limulus test, and TNF- ⁇ was assayed by ELISA.
  • Heat-killed E. coli has no proliferative activity, but it contains large amount of LPS and CpG DNA, which can simulate the stimulation of LPS and CpG DNA in vivo.
  • the LPS and TNF- ⁇ level in mice blood of heat-killed E. coli control group began to increase rapidly 4 h after injection, and fell back to initial state within 24 to 48 h.
  • the LPS and TNF- ⁇ level in mice blood of KA or KB treatment group at various time points were significantly decreased (p ⁇ 0.05 or p ⁇ 0.01).
  • KB was separately diluted with PBS to make solutions of 0.25, 0.5, 1, 2 and 4 ⁇ M; 5 ⁇ l of each solution was separately loaded; binding reaction of KB of various concentration with LPS and CpG DNA was assayed according to the methods in embodiment 1.
  • the dissociation equilibrium constant (K D ) of KB with LPS and CpG DNA was calculated using the IAsys FASTfit software.
  • the dissociation equilibrium constant (K D ) of KB with LPS and CpG DNA is respectively 1.24 ⁇ M and 0.66 ⁇ M.
  • the results were shown in Table 3.
  • CpG DNA 0.0419045 ⁇ 0.0031342 63119.0 ⁇ 672.4 6.63897 ⁇ 10 -7 M
  • KB and PMB were separately diluted with PBS to make a 4 ⁇ M solution; 5 ⁇ l of each solution was loaded respectively; affinity of KB and PMB with LPS and CpG DNA was respectively assayed according to the methods in embodiment 1.
  • PMB is an antagonistic drug of LPS.
  • PMB has high affinity with LPS, and its binding force with LPS is almost 2 times as much as that of KB. But PMB almost has no binding effects on CpG DNA.
  • KB has high affinity with CpG DNA, which suggests that KB can bind to both LPS and CpG DNA.
  • Figure 14a shows the affinity detection of KB and PMB with LPS
  • Figure 14b shows the affinity detection of KB and PMB with CpG DNA.
  • KB and PMB were separately diluted with LPS-free water to make solutions of 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 ⁇ M; according to the methods in embodiment 4, above solutions were separately mixed with equal volume of LPS (2 ng/ml), and neutralizing activity was assayed.
  • KB was dissolved in DMEM supplemented with 10%(v/v) NCS, subsequently transferred into Culture medium of RAW264.7 cells to achieved final concentrations of 100 and 200 ⁇ M, added with LPS (final concentration of 100 ng/ml) and CpG DNA (final concentration of 10 ⁇ g /ml), incubated for 24 h, and then collected supernatant; the concentration of TNF- ⁇ and IL-6 of each group was detected according to the manufacturer's instructions of ELISA kit; result was expressed by means of mean ⁇ standard deviation.
  • KB can inhibit the release of TNF- ⁇ and IL-6 induced by both LPS and CpG DNA.
  • Figure 16 shows the inhibition of KB on the release of TNF- ⁇ in RAW264.7 cells stimulated by LPS and CpG DNA
  • Figure 16b shows the inhibition of KB on the release of IL-6 in RAW264.7 cells stimulated by LPS and CpG DNA.
  • KB and PMB were separately dissolved in DMEM supplemented with 10%(v/v) NCS, subsequently transferred into culture medium of RAW264.7 cells to achieved final concentrations of 50, 100 and 200 ⁇ M, and then added with LPS (final concentration of 100 ng/ml) and CpG DNA (final concentration of 10 ⁇ g /ml); RAW264.7 cells continued to be incubated; the supernatant were collected four hours later for TNF- ⁇ detection; the supernatant were collected 12 hours later for IL-6 detection; the concentration of TNF- ⁇ and IL-6 of each group was detected according to the manufacturer's instructions of ELISA kit; result was expressed by means of mean ⁇ standard deviation.
  • KB can inhibit the release of TNF- ⁇ and IL-6 induced by both LPS and CpG DNA, and PMB can only inhibit the release of TNF- ⁇ and IL-6 induced by LPS.
  • Figure 17a shows the inhibition of KB and PMB on the release of TNF- ⁇ in RAW264.7 cells stimulated by LPS
  • Figure 17b shows the inhibition of KB and PMB on the release of TNF- ⁇ in RAW264.7 cells stimulated by CpG DNA
  • Figure 17c shows the inhibition of KB and PMB on the release of IL-6 in RAW264.7 cells stimulated by LPS
  • Figure 17d shows the inhibition of KB and PMB on the release of IL-6 in RAW264.7 cells stimulated by CpG DNA.
  • KM mice were killed by cervical dislocation and immediately immersed in 75% ethanol for skin degerming; then abdominal skin was aseptically cut open; precooling DMEM cell culture medium was slowly injected in exposed peritoneum with a 5 ml syringe; murine abdomen was gently massaged for sufficient cell collection; subsequently, DMEM was withdrew, transferred into 10 ml centrifuge tube, centrifuged at 500 rpm for 5 min, resuspended in DMEM supplemented with 10%(v/v) NCS, transferred into cell culture bottle, and then cultured at 37 °C in a 5% CO 2 humidified incubator for 2 h; culture medium was replaced with fresh culture medium to remove unattached cells; over 95% of the remained cells were murine peritoneal macrophages, which continued to be cultured and proliferated.
  • KB and PMB were separately diluted in DMEM supplemented with 10%(v/v) NCS and transferred into culture medium of RAW264.7 cells to achieved final concentrations of 50, 100 and 200 ⁇ M; RAW264.7 cells continued to be incubated; the supernatant was collected four hours later for TNF- ⁇ detection; the supernatant at were collected 12 hours later for IL-6 detection; the concentration of TNF- ⁇ and IL-6 of each group was detected according to the manufacturer's instructions of ELISA kit; result was expressed by means of mean ⁇ standard deviation.
  • Figure 18a shows the inhibition of KB and PMB on the release of TNF- ⁇ in murine peritoneal macrophages stimulated by LPS
  • Figure 18b shows the inhibition of KB and PMB on the release of TNF- ⁇ in murine peritoneal macrophages stimulated by CpG DNA
  • Figure 18c shows the inhibition of KB and PMB on the release of IL-6 in murine peritoneal macrophages stimulated by LPS
  • Figure 18d shows the inhibition of KB and PMB on the release of IL-6 in murine peritoneal macrophages stimulated by CpG DNA.
  • Reverse-transcription reaction mixture (include: Rnase-free H 2 O, 10 ⁇ l; 5xRT buffer, 4 ⁇ l; dNTP mixture, 2 ⁇ l; RNase inhibitor, 1 ⁇ l; Oligo(dT)20, 1 ⁇ l; RNA, 1 ⁇ l; ReverTra Ace, 1 ⁇ l) was prepared on ice bath, mixed, incubated at 42°C for 1 h and at 99°C for 5 min, stored at -20 °C .
  • Reaction mixture which includes 1.5 ⁇ l cDNA, 10.0 ⁇ l 2 ⁇ SYBR Green Master Mix, 0.5 ⁇ l upstream primer (10 ⁇ M), 0.5 ⁇ l downstream primer (10 ⁇ M) and 7.5 ⁇ l RNase-free H 2 O, was added into 0.2 ml PCR tube.
  • Amplification programs are as follows: Step Temperatures time Velocity Cycles Initial denaturation 95°C 60 sec 1 PCR (Polymerase Chain Reaction) 95°C 30 sec 40 58 °C 30 sec 72 °C 60 sec Melting Curve Assay 95°C 60 sec 1 54°C 60 sec 1 55°C 10 sec 0.05°C/sec 80
  • Results were expressed as CT value, and converted into ratio of ⁇ -actin, the internal reference items, according to 2 - ⁇ CT methods. Result was expressed by means of mean ⁇ standard deviation.
  • Figure 19a shows the inhibition of KB on the mRNA expression of TNF- ⁇ in RAW264.7 cells stimulated by LPS and CpG DNA
  • Figure 19b shows the inhibition of KB on the mRNA expression of IL-6 in RAW264.7 cells stimulated by LPS and CpG DNA
  • Figure 19c shows the inhibition of KB on the mRNA expression of iNOS in RAW264.7 cells stimulated by LPS and CpG DNA
  • Figure 19d shows the inhibition of KB on the mRNA expression of COX-2 in RAW264.7 cells stimulated by LPS and CpG DNA.
  • KB 100, 200 ⁇ M
  • Figure 20 shows the inhibition of KB on the mRNA expression of TNF- ⁇ in murine peritoneal macrophages stimulated by LPS and CpG DNA
  • Figure 20b shows the inhibition of KB on the mRNA expression of IL-6 in murine peritoneal macrophages stimulated by LPS and CpG DNA.
  • RAW264.7 cells was diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS, transferred into 96-well plates (200 ⁇ l per well) and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h.
  • RAW264.7 cells were diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS, transferred into 96-well plates (200 ⁇ l per well) and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h.
  • KB can still inhibit the release of TNF- ⁇ in RAW264.7 cells induced by LPS and CpG DNA in a dose-dependant manner, which eliminated the possibility that KB play an indirect role in inhibition on LPS and CpG DNA by acting on the serum protein.
  • the results were shown in Figure 22 .
  • Figure 22a shows the inhibition of KB on TNF- ⁇ release in RAW264.7 cells induced by LPS and CpG DNA, in which KB was loaded after preincubation with LPS or CpG DNA
  • Figure 22b shows the inhibition of KB on TNF- ⁇ release in RAW264.7 cells induced by LPS and CpG DNA, in which KB was loaded at various time points
  • Figure 22c shows the inhibition of KB on TNF- ⁇ release in RAW264.7 cells induced by LPS and CpG DNA, in which KB was loaded without serum.
  • RAW264.7 cells were stimulated by six pathogen-associated molecular patterns, LPS (100 ng/ml), CpG DNA (CpG, 10 ⁇ g/ml), Pam3CSK4 (Pam3, 10 ⁇ g/ml), Poly I:C (I:C, 20 ⁇ g/ml), TNF- ⁇ (50 ng/ml) and IL-1 ⁇ (50 ng/ml), and added with KB (final concentration of 200 ⁇ M) in the meantime. According to the method in embodiment 6, the supernatant was collected. The concentration of TNF- ⁇ was detected according to the manufacturer's instructions of ELISA kit. Result was expressed by means of mean ⁇ standard deviation.
  • KB (200 ⁇ M) intervention can only inhibit the release of TNF- ⁇ and IL-6 induced by LPS and CpG DNA, but has no antagonistic effect on other irritant.
  • the result showed that KB effect only targets at LPS and CpG DNA.
  • Figure 23 shows the inhibition of KB on TNF- ⁇ release in RAW264.7 cells induced by various pathogen-associated molecular patterns
  • Figure 23b shows the inhibition of KB on IL-6 release in RAW264.7 cells induced by various pathogen-associated molecular patterns.
  • RAW264.7 cells was diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS; 2 ml of the above suspension was added into 24-well cell culture plates and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h; for the purpose of experiment three groups were established: medium group, stimulation group and KB treatment group; no reagent was added into medium group; stimulation group was added with FITC-LPS (200 ng/ml) and 5-FAM-CpG DNA (10 ⁇ g/ml); KB treatment group was added with KB (final concentrations of 50, 100 and 200 ⁇ M) in the meantime of adding LPS and CpG DNA; cells continued to be cultured for 30 min, washed thrice with PBS, and stored in dark place until the fluorescence intensity of cell membrane surfaces was detected by flow cytometry.
  • RAW264.7 cells were cultured in 20 mm cell culture dishes for confocal microscopy applications, and diluted to 5 ⁇ 10 5 /ml in DMEM supplemented with 10% (v/v) NCS; 1 ml of the above suspension was transferred into each cell culture dishes and cultured at 37 °C in a 5% CO 2 humidified incubator for 4 h; for the puepose of experiment three groups were established, medium group, stimulation group and KB treatment group; the concentration and loading patterns of each group were consistent with Embodiment 12; the cells were then fixed with 4% paraform for 10 min after 30 min of culture, and washed thrice with PBS; nucleus were stained with DAPI (100 ng-mL -1 ) for 2 min, washed with PBS thrice, mounted with a solution of 50% glycerol and 50% PBS, and stored in dark place until intensity and distribution of fluorescence of LPS and CpG DNA on RAW 264.7 cells surface were observed under a confocal microscopy
  • Figure 25 shows the KB influence on binding and cellular internalization of LPS to RAW264.7 cells
  • Figure 25b shows the KB influence on mean fluorescence intensity of 5-FAM-CpG DNA on RAW264.7 cell surface.
  • TLR4 and TLR9 are as follows: Sequences Mouse TLR4 Upstream primer: 5'- AAGGCATGGCATGGCTTACAC-3' Downstream primer: 5'-GGCCAATTTTGTCTCCACAGC-3' Mouse TLR9 Upstream primer: 5'-TCGCTCAACAAGTACACGC-3' Downstream primer: 5'-GCTCTGCATCATCTGCCTC-3' Mouse ⁇ -actin Upstream primer: 5'-GGGAAATCGTGCGTGACATCAAAG-3' Downstream primer: 5'-CATACCCAAGAAGGAAGGC TGGAA-3'
  • results of RT-PCR assay show that with the expressions of TLR4 and TLR9 in untreated RAW264.7 cells serving as a control, stimulation of LPS and CpG DNA can significantly up regulated the expression of TLR4 and TLR9; after intervention with 100 and 200 ⁇ M KB, the up-regulated expression of TLR4 and TLR9 was significantly inhibited, which suggested that KB can block the up-regulated expression of TLR4 and TLR9, and then inhibited further stimulation. The results were shown in Figure 26 .
  • Figure 26a shows the inhibition of KB on the up-regulated expression of TLR4 mRNA induced by LPS and CpG DNA
  • Figure 26b shows the inhibition of KB on the up-regulated expression of TLR9 mRNA induced by LPS and CpG DNA.
  • RAW264.7 cells were diluted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS; 5 ml of the above suspension was transferred into cell culture bottles and cultured at 37 °C in a 5% CO2 humidified incubator for 4 h; a preliminary experiment was first performed, in which RAW264.7 cells were stimulated by LPS (100 ng/ml), CpG DNA (10 ⁇ g/ml), TNF- ⁇ (50 ng/ml) or IL-1 ⁇ (50 ng/ml) for 15, 30, 45 and 60 min respectively; for the purpose of formal experiment a medium group, a KB control group, stimulation group (LPS, CpG DNA, TNF- ⁇ or IL-1 ⁇ ) and a KB treatment group (LPS+KB group, CpG DNA+KB group, TNF- ⁇ +KB group and IL-1 ⁇ +KB group) were established; no reagent was added in medium group; KB control group was only added with KB
  • the detection results of I ⁇ B- ⁇ show that: the expression levels of p38 and tubulin internal reference in each group were basically in agreement; with medium group serving as a control, addition of KB alone had no influence on the expression of I ⁇ B- ⁇ and p-I ⁇ B- ⁇ ; stimulation of LPS and CpG DNA lead to the degradation of I ⁇ B- ⁇ (the degradation of I ⁇ B- ⁇ can not be detected after RAW264.7 cells were stimulated by LPS and CpG DNA for more than 45 min, therefore the stimulation time in formal experiment was chosen as 30 min) and the up-regulated expression of p-I ⁇ B- ⁇ ; after intervention by KB at the same concentration, the degradation of I ⁇ B- ⁇ and the up-regulated expression of p-I ⁇ B- ⁇ both were significantly inhibited, which suggested that KB can neutralized LPS and CpG DNA and inhibited the activation of intracellular signaling molecules induced by them.
  • Figure 27 shows the degradation of I ⁇ B- ⁇ in RAW264.7 cells after stimulated by LPS, CpG DNA, TNF- ⁇ and IL-1 ⁇ for different time (15, 30, 45, and 60 mins), and Figure 27b shows the inhibition of KB on up-regulated phosphorylation of signaling molecules p38 induced by LPS, CpG DNA, TNF- ⁇ and IL-1 ⁇ , and the inhibition of KB on degradation and phosphorylation of I ⁇ B- ⁇ , in RAW264.7 cells.
  • NF- ⁇ B Transcription factors such as NF- ⁇ B were activated by activation of signaling molecules induced by LPS and CpG DNA.
  • P50 and p65 the subunit of NF- ⁇ B in nuclear proteins, were detected using ELISA.
  • the results show that: As compared with the unstimulated RAW264.7 cells, KB alone has no influence on the expression of p50 and p65; the stimulation of LPS and CpG DNA can lead to a significant increase of p50 and p65 levels; after intervention of 200 ⁇ M KB, the increase of p50 and p65 levels in nuclear proteins was significantly inhibited.
  • Results of luciferase reporter gene assay also show that KB can inhibit NF- ⁇ B activation in RAW264.7 cells induced by LPS and CpG DNA.
  • the results were shown in Figure 28 .
  • Figure 28a shows the inhibition of KB on up-regulation of NF- ⁇ B p50 subunit in RAW264.7 cell nucleus induced by LPS and CpG DNA
  • Figure 28b shows the inhibition of KB on up-regulation of NF- ⁇ B p65 subunit in RAW264.7 cell nucleus induced by LPS and CpG DNA
  • Figure 28c shows assay of KB inhibition on NF- ⁇ B activation in RAW264.7 cells induced by LPS and CpG DNA using a luciferase reporter gene assay.
  • RAW264.7 cells was adjusted to 1 ⁇ 10 6 /ml in DMEM supplemented with 10% (v/v) NCS; for the purpose of experiment a LPS or CpG DNA stimulation group, a KB treatment group and a medium group were established; LPS or CpG DNA stimulation group was separately added with LPS (100 ng/ml) or CpG DNA (10 ⁇ g/ml); KB treatment group was added with KB (200 ⁇ M) in the meantime of adding LPS (100 ng/ml) or CpG DNA (10 ⁇ g/ml); no reagent was added in medium group; cells continued to be cultured for 4 h after sample loading; total RNA extraction and reverse transcription were performed according to the procedure of embodiment 15; mRNA of TLR
  • Reaction mixtures were added into 0.2 ml PCR tubes.
  • the reaction mixture included 1 ⁇ l cDNA, 10.0 ⁇ l 2 ⁇ Taq Master Mix, 1 ⁇ l upstream primer (10 ⁇ M), 1 ⁇ l downstream primer (10 ⁇ M) and 7 ⁇ l RNase-free H 2 O.
  • PCR programs are as follows: Steps Temperature Times Cycles Initial denaturation 94°C 3 min Denaturation 94°C 30 sec Annealing 58°C 30 sec 26 Extension 72°C 1 min Final extension 72°C 7 min Preservation 4°C
  • PCR products were detected by agarose gel electrophoresis; 1% agarose gel was prepared and cast; 5 ⁇ l PCR products of each tube were loaded, electrophoresed at 100 v for 30 min; then gel was took out and scanned; the images were analysed by Quantity One software.
  • Figure 29a shows the inhibition of KB on up-regulated expression of TLR4 and TLR9 mRNA in RAW264.7 cells induced by LPS and CpG DNA
  • Figure 29b shows the inhibition of KB on up-regulated expression of MyD88 mRNA in RAW264.7 cells induced by LPS and CpG DNA
  • Results of western blot show that KB (200 ⁇ M) can inhibit the up-regulation of NF- ⁇ B p65 in RAW264.7 cell nucleus induced by LPS (100 ng/ml) and CpG DNA (10 ⁇ g/ml), which suggests that KB can inhibit the activation of NF- ⁇ B in RAW264.7 cells induced by stimulation of LPS and CpG DNA.
  • the results were shown in Figure 29c .
  • Figure 30a shows the influence of KB on vitality of RAW264.7 cells
  • Figure 30b shows the influence of the combination of KB with LPS or CpG DNA on vitality of RAW264.7 cells
  • Figure 30c shows the influence of KB on vitality of murine peritoneal macrophages
  • Figure 30d shows the influence of the combination of KB with LPS or CpG DNA on vitality of murine peritoneal macrophages.
  • Kunming mice were randomly divided into two groups, and there were 56 mice in each group; heat-killed E. coli was diluted to make 1.0 ⁇ 10 9 CFU/ml suspension, and KB was dissolved to 6 mg/ml with sterile saline; caudal vein injection was adoped; medium group was injected with heat-killed Escherichia coli (0.2 ml per 20 g body weight) and sterile saline (0.2 ml per 20 g body weight); KB treatment groups were injected with heat-killed E. coli suspension S (0.2 ml per 20 g body weight) and KB (0.2 ml per 20 g body weight).
  • LPS levels in normal KM mice plasma were below the detection limit (less than 0.0015 EU/ml); after injection of sublethal dose of heat-killed E. coli (1.0 ⁇ 10 10 CFU/ml), LPS levels in KM mice plasma increased rapidly, peaked at 8 h (819.42 ⁇ 159.02 EU/ml), then decreased gradually and close to normal levels at 72 h; the change tendency of LPS levels in mice plasma in KB treatment group were consistent with heat-killed E.coli control group in this period, and LPS levels in mice plasma in KB treatment group were significantly lower (p ⁇ 0.05 or p ⁇ 0.01) than heat-killed E.coli control group in time point of 4, 8, 12, 24 and 48 h; there were only basic levels of TNF- ⁇ exist in normal KM mice serum (less than 100 pg/ml); after injection of sublethal dose of heat-killed E.
  • Figure 32a shows the influence of KB on LPS levels in plasma of mice challenged by sublethal dose of heat-killed Escherichia coli.
  • Figure 32b shows the influence of KB on TNF- ⁇ levels in serum of mice challenged by sublethal dose of heat-killed Escherichia coli.
  • KB was diluted to 6.0 mg/ml with sterile saline, and heat-killed E. coli suspension was diluted to 1.0 ⁇ 10 10 CFU/ml with sterile saline for further detection; a total of 96 Kunming mice, half male and half female, were randomly divided into heat-killed E. coli control group, 0 h KB treatment group, 2 h KB treatment group, 4 h KB treatment group, 6 h KB treatment group and 8 h KB treatment group; each group has 16 mice; after animal weighing, heat-killed E.
  • mice in each group were housed separately and feed with sufficient and equivalent food and water.
  • the general status (mental status, appetite, activity and response to stimuli), mortality rate and time of death, of mice in each group, were observed in 7 days after injection.
  • a total of 16 Kunming mice were randomly divided into four groups; each group has 4 mice, half male and half female; for the purpose of experiment a medium group and a KB (60 mg/kg) treatment group were established; mice in medium group were immediately killed by cervical dislocation, and KB treatment group were killed separately killed at 24, 48 and 72 h after injection; the cavitas thoracis and abdominal cavity of mice were cut open; organs such as heart, liver, lung, kidney and intestine were moved out, rinsed with sterile saline, plunged into 10% formaldehyde solution for fixing, dehydrated, embedded in paraffin, HE dyed and mounted; organ histopathological changes were observed under a light microscope.
  • Figure 34 shows the lung morphology of mice after KB injection
  • Figure 34b shows the liver morphology of mice after KB injection
  • Figure 34c shows the kidney morphology of mice after KB injection
  • Figure 34d shows the cardiac muscle morphology of mice after KB injection.

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Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101829075B (zh) * 2010-04-27 2011-07-20 中国人民解放军第三军医大学第一附属医院 苦柯胺a和苦柯胺b的用途
CN102464591B (zh) * 2010-11-10 2016-08-24 中国人民解放军第三军医大学第一附属医院 苦柯胺b盐及制备方法和用途
CN102267922B (zh) * 2011-04-27 2014-11-19 中国人民解放军第三军医大学第一附属医院 一类多胺化合物及制备方法和用途
CN102641240B (zh) * 2012-05-22 2014-07-16 天津红日药业股份有限公司 一种苦柯胺b盐注射剂及其制备方法
BR112016008328B1 (pt) 2013-10-17 2022-04-12 Swimc Llc Artigo revestido
CN103769060B (zh) * 2014-01-23 2015-10-21 重庆安体新生物技术有限公司 用于清除细菌内毒素、dna和肽聚糖的吸附剂和制备方法及用途
BR112016026342B1 (pt) 2014-05-19 2022-03-03 Swimc Llc Artigo
CN104725278B (zh) * 2015-01-23 2017-01-25 天津红日药业股份有限公司 一种苦柯胺b中间体的制备方法
CN105301157B (zh) * 2015-10-12 2017-03-29 天津红日药业股份有限公司 一种甲磺酸苦柯胺b有关物质检测方法
CN105277721B (zh) * 2015-10-14 2017-10-13 广州白云山和记黄埔中药有限公司 一种鉴别口炎清制剂原料山银花和金银花的方法
CN105348137B (zh) * 2015-10-29 2018-06-12 重庆安体新生物技术有限公司 多胺衍生物药用盐及制备方法和用途
CN109772269B (zh) * 2017-11-13 2022-06-21 天津红日药业股份有限公司 一种内毒素吸附剂及其制备方法和用途
KR102156540B1 (ko) * 2018-04-11 2020-09-17 한양대학교 에리카산학협력단 구꼬아민 a 및 구꼬아민 b를 유효성분으로 함유하는 골격근 위축 예방 또는 개선용 조성물
WO2019198982A1 (ko) * 2018-04-11 2019-10-17 한양대학교 에리카산학협력단 구꼬아민 a 및 구꼬아민 b를 유효성분으로 함유하는 골격근 위축 예방 또는 개선용 조성물
KR102171518B1 (ko) * 2019-02-21 2020-10-29 한양대학교 에리카산학협력단 지골피 추출물을 유효성분으로 함유하는 근 위축 예방 또는 치료용 조성물
CN110302282A (zh) * 2019-06-06 2019-10-08 中国人民解放军南部战区总医院 败酱草提取物在制备抗耐药鲍曼不动杆菌药物中的应用
US20220248732A1 (en) * 2021-02-05 2022-08-11 Frito-Lay North America, Inc. Saltiness-Perception Enhancing Compounds
CN113112494A (zh) * 2021-04-29 2021-07-13 同济大学 一种icf热斑空间形貌随时间演化图像的三维协同诊断装置
CN114053255A (zh) * 2021-12-24 2022-02-18 天津红日药业股份有限公司 甲磺酸苦柯胺b在制备治疗脓毒症休克药物中的应用
CN114773218B (zh) * 2022-06-02 2024-06-04 云南省药物研究所 一种地骨皮乙素的制备方法
CN115820757B (zh) * 2022-11-29 2023-06-20 中国科学院华南植物园 枸杞bahd酰基转移酶的编码基因和蛋白的应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1133278A2 (en) * 1999-07-07 2001-09-19 Her Majesty The Queen As Represented By The Minister Of National Defence Of Her Majesty's Canadian Government Delivery of liposomal-encapsulated antioxidants and applications thereof
JP4010714B2 (ja) * 1999-09-29 2007-11-21 独立行政法人科学技術振興機構 イリドイド誘導体とこれを有効成分とする抗炎症剤並びに自己免疫疾患剤
EP1581243A4 (en) * 2002-12-06 2008-01-02 Agennix Inc ORAL LACTOFERRINE FOR THE TREATMENT OF SEPSIES
CN1953761A (zh) * 2004-02-25 2007-04-25 港大科桥有限公司 使用宁夏枸杞提取物作为视网膜神经节细胞变性的神经保护剂的方法
CN100409864C (zh) * 2005-05-18 2008-08-13 中国人民解放军第三军医大学第一附属医院 一种从中草药中筛选、分离的活性组分或活性物质在制备治疗脓毒症药物中的应用
JP2007051103A (ja) * 2005-08-19 2007-03-01 Taiyo Kagaku Co Ltd 抗酸化組成物
ES2434039T3 (es) * 2006-07-24 2013-12-13 Yeda Research And Development Co. Ltd. Composiciones farmacéuticas que comprenden ccl2 para su uso en el tratamiento de la inflamación
US20100273868A1 (en) * 2007-01-05 2010-10-28 Cornerstone Therapeutics Inc. R-Zileuton for Use in Conditions Associated with Increased 5-Lipoxygenase and/or Leukotriene Activity (EG Asthma)
CN101829075B (zh) * 2010-04-27 2011-07-20 中国人民解放军第三军医大学第一附属医院 苦柯胺a和苦柯胺b的用途
CN102464591B (zh) 2010-11-10 2016-08-24 中国人民解放军第三军医大学第一附属医院 苦柯胺b盐及制备方法和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAJANAN SHERBET: "Bacterial infections and the pathogenesis of autoimmune conditions", BRITISH JOURNAL OF MEDICAL PRACTITIONERS, 1 March 2009 (2009-03-01), pages 6, XP055182491, Retrieved from the Internet <URL:http://www.bjmp.org/files/march2009/bjmp0309sherbet.pdf> *
HADJIPAVLOU-LITINA DIMITRA ET AL: "Kukoamine A analogs with lipoxygenase inhibitory activity.", JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY OCT 2009, vol. 24, no. 5, October 2009 (2009-10-01), pages 1188 - 1193, XP008172270, ISSN: 1475-6374 *

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CN101829075A (zh) 2010-09-15
WO2011134271A1 (zh) 2011-11-03
CA2800891A1 (en) 2011-11-03
EP2591776A4 (en) 2013-08-14
US20170189355A1 (en) 2017-07-06
JP2013525382A (ja) 2013-06-20
CN101829075B (zh) 2011-07-20
EP2591776A1 (en) 2013-05-15
US10039729B2 (en) 2018-08-07

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